Alveolar rhabdomyosarcoma comprises a rare highly malignant tumor presumed to be associated with skeletal muscle lineage in children. The hallmark of the majority of alveolar rhabdomyosarcoma is a chromosomal translocation that generates the PAX3-FOXO1 fusion protein, which is an oncogenic transcription factor responsible for the development of the malignant phenotype of this tumor. Alveolar rhabdomyosarcoma cells are dependent on the oncogenic activity of PAX3-FOXO1, and its expression status in alveolar rhabdomyosarcoma tumors correlates with worst patient outcome, suggesting that blocking this activity of PAX3-FOXO1 may be an attractive therapeutic strategy against this fusion-positive disease. In this study, we screened small molecule chemical libraries for inhibitors of PAX3-FOXO1 transcriptional activity using a cell-based readout system. We identified the Sarco/endoplasmic reticulum Ca2+-ATPases (SERCA) inhibitor thapsigargin as an effective inhibitor of PAX3-FOXO1. Subsequent experiments in alveolar rhabdomyosarcoma cells showed that activation of AKT by thapsigargin inhibited PAX3-FOXO1 activity via phosphorylation. Moreover, this AKT activation appears to be associated with the effects of thapsigargin on intracellular calcium levels. Furthermore, thapsigargin inhibited the binding of PAX3-FOXO1 to target genes and subsequently promoted its proteasomal degradation. In addition, thapsigargin treatment decreases the growth and invasive capacity of alveolar rhabdomyosarcoma cells while inducing apoptosis in vitro. Finally, thapsigargin can suppress the growth of an alveolar rhabdomyosarcoma xenograft tumor in vivo. These data reveal that thapsigargin-induced activation of AKT is an effective mechanism to inhibit PAX3-FOXO1 and a potential agent for targeted therapy against alveolar rhabdomyosarcoma. Mol Cancer Ther; 12(12); 2663–74. ©2013 AACR.
Alveolar rhabdomyosarcoma is an aggressive form of soft-tissue pediatric sarcoma and exhibits a poor prognosis (1). The overall 5-year survival rate for patients with alveolar rhabdomyosarcoma is about 50% (2). Despite modern multimodal treatment approaches, the outcome for patients with alveolar rhabdomyosarcoma remains poor. This highlights the need for new therapeutic strategies to improve alveolar rhabdomyosarcoma patients' outcome.
The majority of alveolar rhabdomyosarcoma is characterized by a specific chromosomal translocation t(2;13) that leads to the expression of the novel chimeric transcription factor PAX3-FOXO1. The PAX3-FOXO1 protein contains the DNA-binding portions of PAX3 fused to the transactivation domain of FOXO1 (3) and acts as a potent transcriptional activator (4). Its transcriptional activity influences the expression of genes that affect cell growth, motility, and apoptosis (5), and forced expression of PAX3-FOXO1 alone can induce oncogenic transformation (6). However, PAX3-FOXO1 needs additional genetic lesions to give rise to alveolar rhabdomyosarcoma (7–9), which is also supported by mouse models of alveolar rhabdomyosarcoma (10). The oncogenic role of PAX3-FOXO1, which is believed to be attributed by its transcriptional activity (5, 11), is necessary for the maintenance of the transformed phenotype (12–14). Besides the oncogenic role of PAX3-FOXO1 in alveolar rhabdomyosarcoma pathogenesis, its prognostic significance has also been acknowledged in patients with this fusion-positive disease where PAX3-FOXO1–positive tumors are associated with an aggressive phenotype and significantly worse prognosis (15–17). Therefore, these evidences suggest that the inhibition of PAX3-FOXO1 activity may provide an effective treatment strategy against fusion-positive alveolar rhabdomyosarcoma. Indeed, a variety of approaches have been taken to inhibit PAX3-FOXO1 (12, 18–20); however, thus far, no pharmaceuticals have been developed against this fusion oncoprotein.
This study described the development of a screening approach to identify small molecule inhibitors of PAX3-FOXO1 transcriptional activity. To this end, a PAX3-FOXO1–responsive luciferase reporter was used as a primary readout followed by several secondary assays in evaluating PAX3-FOXO1 activity in alveolar rhabdomyosarcoma cells to evaluate 3,280 pharmacological compounds. The most active candidate of PAX3-FOXO1 inhibitor identified in this screen was thapsigargin that acts as a potent inhibitor of Sarco/endoplasmic reticulum Ca2+-ATPases (SERCA; ref. 21). Further characterization of this compound revealed that activation of AKT by thapsigargin inhibited the transcriptional activity of PAX3-FOXO1 via phosphorylation and subsequently promoted proteasomal degradation. This thapsigargin-induced AKT activation was apparently due to the increased intracellular Ca2+ levels. Moreover, thapsigargin inhibits the malignant characteristics of alveolar rhabdomyosarcoma cells in vitro and blocks alveolar rhabdomyosarcoma tumor growth in vivo. These findings offer a novel preclinical rationale for a thapsigargin-based approach to target PAX3-FOXO1 activity as a therapeutic strategy against alveolar rhabdomyosarcoma.
Materials and Methods
Cells and cell culture
Mouse alveolar rhabdomyosarcoma cell lines U20497T and U20325 were previously described (22, 23). Human alveolar rhabdomyosarcoma cell lines Rh7, Rh28, Rh30, and Rh41 were provided by Dr. Peter. J. Houghton (Nationwide Children's Hospital, OH). C2C12, RD, 293A, and Phoenix-Ampho cell lines were authenticated as described previously (23, 24). U20497T-PRSLuc and U20325-PRSLuc cells expressing the PAX3-FOXO1–responsive reporter luciferase (6XPRS-Luc) generated through lentivirus transduction were described previously (23). A similar strategy was applied to generate Rh30-PRSLuc and RD-PRSLuc reporter cells and CMV-driven luciferase reporter U20325-CMVLuc cells. The alveolar rhabdomyosarcoma cells were authenticated by PAX3-FOXO1 expression, a hallmark of these cells. Cell lines used in this study were not propagated more than 2 months and/or 10 passages. All these cells were grown in Dulbecco's Modified Eagle Medium (DMEM) with 10% FBS except C2C12 cells (20% FBS). Additional details, including reagents, inhibitors, plasmids, antibodies, and virus production and transduction, are described in Supplementary Materials and Methods.
Screening of small molecule libraries
To screen for inhibitors of the PAX3-FOXO1 transcriptional activity, a total of 3,280 compounds were used. U20325-PRSLuc cells were used as a screening readout to identify compounds that decreased reporter luciferase activity. Briefly, the above cells were incubated with library compounds for 24 hours followed by luciferase activity assay. The criterion used to define hits in the primary screen was the decreased luciferase activity greater than 70% of solvent dimethyl sulfoxide (DMSO) control. Compounds fulfilling the above criterion were subsequently subjected to a secondary screen to evaluate compounds that decreased luciferase activity in a dose-response manner but had no effect on cell viability. Nine compounds were selected on the basis of dose-dependent decreased luciferase activity, while maintaining greater than 80% cell viability. Details of small molecule libraries and screening of primary and secondary assays are provided in Supplementary Materials and Methods.
Luciferase assay, immunoblot, and real-time reverse transcription PCR analysis
Luciferase assay and analysis of immunoblot and real-time PCR (qRT-PCR) were done as described in Supplementary Materials and Methods.
Measurement of intracellular calcium
Intracellular free calcium in alveolar rhabdomyosarcoma cells was determined using Fluo-4 Direct Calcium Assay kit (Invitrogen) according to the Manufacturer's protocol. In brief, 2 × 104 cells per well in 96-well plate were incubated with Fluo-4 Direct calcium reagent loading solution and incubated for 30 minutes at 37°C followed by 30 minutes at room temperature. The cells were then stimulated by the addition of thapsigargin, and cytosolic calcium was measured by monitoring fluorescence intensity at 516 nm, after excitation at 494 nm.
Chromatin immunoprecipitation, cell proliferation, clonogenic, and soft agar colony formation assays
These assays were conducted as described previously (23, 24). Additional details are described in Supplementary Materials and Methods.
Apoptosis DNA ladder formation assay
Rh28 and Rh30 cells (5 × 106) were seeded in 10-cm2 dishes and incubated overnight. Cells were then treated with thapsigargin or vehicle control. After 4-day treatment, both attached and floated cells were collected, washed, and lysed by cell lysis buffer (25). Cell extracts were clarified by centrifuge and SDS was added to the extracts (1% final). The extracts were then treated with RNase A (5 μg/μL) for 2 hours at 56°C followed by proteinase K (2.5 μg/μL) for 2 hours at 37°C. Subsequently, DNA was extracted by with a mixture of phenol, chloroform isoamyl alcohol followed by ethanol precipitation and resolved on a 2% agarose gel and documented.
This assay was conducted according to manufacturer's protocol (BD Bioscience). Briefly, DMEM containing 10% FBS was added to the lower chamber in a Matrigel chamber containing 24-well plate. Rh30 and U20325 cells (2 × 104 cells per well) treated with thapsigargin or vehicle control were suspended in DMEM containing 1% FBS and added to the top of each insert well followed by incubation at 37°C for 24 hours. Following incubation, the noninvading cells were removed by scrubbing from the upper surface of the insert. The cells on the lower surface of the insert were stained with Hoechst dye, visualized, and analyzed.
All animal studies were conducted in accordance with the Guidance for the Care and Use of Laboratory Animals (NIH) and approved by the Institutional Animal Care and Use Committee. Details of the xenograft experiments are described in Supplementary Materials and Methods. Briefly, 5 × 106 Rh28 cells were injected into flank of 8- to 10-week-old NOG mice. Once tumor volume reached a minimum 200 mm3, mice were treated via intravenous injection (single treatment) with thapsigargin or PBS control. Tumor volume was measured every 3 days and determined by the formula (W2 × L)/2 where W = width and L = length.
Briefly, formalin-fixed dissected tumors were embedded in paraffin for immunohistochemical analysis. Paraffin sections were the stained with hematoxylin and eosin (H&E) or subjected to immunohistochemical staining for Ki-67 and activated caspase-3. Stained slides were counterstained with hematoxylin, mounted, and images were captured. Additional details are described in Supplementary Materials and Methods.
Unpaired two-tailed Student t-test analyses were conducted to ascertain statistical significance, and P < 0.05 was considered statistically significant.
Screening of small molecule libraries identifies thapsigargin as an inhibitor of PAX3-FOXO1
This study was aimed to identify compounds that inhibit PAX3-FOXO1 activity in alveolar rhabdomyosarcoma cells. A screening was conducted of small molecule libraries that consisted of 3,280 compounds to identify those that suppress PAX3-FOXO1 transcriptional activity. Figure 1A depicted the outline of screening design that identified PFI-6 (thapsigargin) as an inhibitor of PAX3-FOXO1 activity. In the primary screen, the library compounds were evaluated using PAX3-FOXO1–responsive luciferase reporter ARMS-325-6XPRS-Luc cells (23), described hereafter as U20325-PRSLuc, to monitor PAX3-FOXO1 activity. The increased luciferase activity observed in these cells was abolished by PAX3-FOXO1 shRNA (23), which also depleted the PAX3-FOXO1 protein (Supplementary Fig. S1A). However, such effects were not observed with scrambled control shRNA, validating PAX3-FOXO1–mediated increase in luciferase activity in U20325-PRSLuc cells. Most PAX3-FOXO1–positive human alveolar rhabdomyosarcoma cells, including Rh28 and Rh30 cells, express a low level of wild-type PAX3 (ref. 26; Supplementary Fig. S1B). As PAX3-FOXO1 contains the DNA-binding regions of PAX3 (5), its presence in human alveolar rhabdomyosarcoma cells would interfere in the identification of inhibitors of PAX3-FOXO1 activity. In contrast, mouse U20325 alveolar rhabdomyosarcoma cells (27) express a typical fusion PAX3-FOXO1, myogenic MyoD proteins, no wild-type PAX3 (Supplementary Fig. S1C and S1D). Therefore, the basis of using U20325-PRSLuc cells was that wild-type PAX3 would not interfere in identifying compounds that inhibit PAX3-FOXO1–mediated reporter luciferase activity in these cells. In the primary screen, “hit” compounds were selected upon treatment of U20325-PRSLuc cells with library compounds based on more than 70% inhibition of luciferase activity. Hit compounds were validated with secondary screening, which included a dose–response assay and parallel cell viability measurement in reporter cells. The secondary screen yielded 9 compounds (cataloged as PFI 1 to 9) that exhibit dose-dependent inhibition of luciferase activity with insignificant cell cytotoxicity (Supplementary Fig. S2A). These compounds were then subjected to additional filters to eliminate compounds that either inhibitors of general transcription or wild-type PAX3. General transcriptional inhibition was tested using U20325 cells expressing a constitutively active CMV-driven reporter luciferase. Inhibition of PAX3 was evaluated in PAX3-FOXO1–negative RD-PRSLuc cells, which are ERMS subtype RD cells that express endogenous PAX3 and the 6XPRS-luc reporter gene (Supplementary Fig. S2B). After filtering, 4 compounds remained, which were further characterized using 2 additional Rh30-PRSLuc and U20497T-PRSLuc (23) reporter cells. Of the 4 compounds, 3 showed the inhibitory effect on the luciferase activity, with PF6 being the most potent (Supplementary Fig. S2C). This compound was identified as SERCA inhibitor thapsigargin. Its inhibitory effect on PAX3-FOXO1–responsive luciferase activity was re-evaluated in parallel both in human (Rh30-PRSLuc) and mouse (U20325-PRSLuc and U20497T-PRSLuc) cells. As shown in Fig. 1B, thapsigargin severely inhibits luciferase activity in these cells. The IC50 value of thapsigargin inhibited luciferase activity in U20325-PRSLuc cells was determined as 2.3 nmol/L (Fig. 1C). Such IC50 value of thapsigargin determined in Rh30PRSLuc cells was 8.0 nmol/L (data not shown). Because thapsigargin inhibits PAX3-FOXO1–driven reporter transcription, the effect of thapsigargin on the expression status of PAX3-FOXO1 was evaluated using Rh28 and Rh30 cells. As shown in Fig. 1D, thapsigargin did not suppress PAX3-FOXO1 mRNA levels. Therefore, the effect of thapsigargin on PAX3-FOXO1 protein levels was determined in a panel of PAX3-FOXO1–expressing alveolar rhabdomyosarcoma cell lines (23, 28). The data showed that thapsigargin decreases PAX3-FOXO1 protein levels in these cells (Fig. 1E). Moreover, thapsigargin caused a decrease in the expression of MyoD, which is a PAX3-FOXO1 downstream target gene (29). In addition, the quantitative RT-PCR analysis confirmed the decreased expression of both MYOD and SKP2, another downstream target of PAX3-FOXO1 (30), in thapsigargin-treated Rh30 cells (Fig. 1F). Thus, thapsigargin exerts its effects on PAX3-FOXO1–dependent gene transcription in alveolar rhabdomyosarcoma cells, in part, by declining PAX3-FOXO1 protein but not mRNA levels.
Thapsigargin-induced AKT activity causes transcriptional inactivation of PAX3-FOXO1 via phosphorylation
Phosphorylation modulates PAX3-FOXO1 transcriptional activity (18, 23, 31), specifically, acute AKT activation inhibits PAX3-FOXO1–dependent gene transcription by inducing its phosphorylation (23). As thapsigargin stimulates activation of AKT (32), phosphorylation status of AKT at Ser437 (as a measurement of AKT activation) and PAX3-FOXO1 was evaluated in Rh30 cells treated with thapsigargin for 2, 4, 6, 8, and 12 hours. Increased AKT activation was detected in thapsigargin-treated cells as early as 2 hours but decreased at the latter 2 time points (Fig. 2A). In addition, an increased level of phosphorylated PAX3-FOXO1 was predominant in thapsigargin-treated cells at 2, 4, and 6 hours, but its level declined with decreased levels of activated AKT at latter time points. The thapsigargin-induced AKT activation coupled with increased levels of phosphorylated PAX3-FOXO1 was correlated with a gradual decrease in luciferase activity in Rh30-PRSLuc cells treated with thapsigargin for same time points (Fig. 2B). The data further showed that the decreased levels of PAX3-FOXO1 at latter time points was associated with decrease in luciferase activity in Rh30PRSLuc cells treated with thapsigargin. The role of thapsigargin-induced AKT activation in modulating PAX3-FOXO1 activity was further characterized in Rh30-PRSLuc cells incubated with thapsigargin in the presence or absence of the AKT kinase inhibitor MK-2206. The data showed that whereas thapsigargin alone decreases PAX3-FOXO1–mediated reporter luciferase activity, MK-2206 abolishes this thapsigargin effect (Fig. 2C). Immunoblot confirmed that thapsigargin-induced AKT activation in these cells was blocked by MK-2206. Next, the effect of thapsigargin-induced AKT activation on PAX3-FOXO1 phosphorylation status was examined by repeating the above experiment in Rh28 and Rh30 cells. The data showed that the levels of phosphorylated PAX3-FOXO1 induced by thapsigargin decreased with MK-2206 co-incubation (Fig. 2D). Finally, to verify that the inhibition of PAX3-FOXO1 activity by thapsigargin was due to AKT activation, luciferase activity in Rh30-PRSLuc cells was determined following the expression of a constitutive active form of AKT (myrAKT-HA). Indeed, constitutive active AKT retrovirus but not control retrovirus decreases luciferase activity (Fig. 2E). Together, the results suggest that thapsigargin inhibited the transcriptional activity of PAX3-FOXO1 via phosphorylation due to an increase in the levels of activated AKT.
Thapsigargin-induced Ca2+ levels correlate with AKT activation inhibited PAX3-FOXO1 transcriptional activity followed by its proteasomal degradation
Thapsigargin disrupts the endoplasmic reticulum (ER) Ca2+ store and elevates cytosolic Ca2+ levels, which affects downstream signaling pathways involved in multiple cellular responses (21, 32, 33). As thapsigargin-activated AKT inhibits PAX3-FOXO1 transcriptional activity in alveolar rhabdomyosarcoma cells (Fig. 2), it was possible that the activation of AKT and subsequent inhibition of PAX3-FOXO1 activity was caused by thapsigargin-induced increase of cytosolic Ca2+ levels in these cells. Indeed, thapsigargin treatment resulted in an elevation of cytosolic Ca2+ levels in Rh28 and Rh30 cells (Fig. 3A). Furthermore, CaCl2 treatment caused an increase in AKT activation in above alveolar rhabdomyosarcoma cells (Fig. 3B) and a decrease in PAX3-FOXO1–responsive reporter luciferase activity in Rh30-PRSLuc cells (Fig. 3C). These results suggest that the effect of thapsigargin on AKT activation and PAX3-FOXO1 transcriptional activity is at least partially due to its effect on intracellular calcium levels.
Although thapsigargin-induced AKT activation correlated with increased levels of phosphorylated PAX3-FOXO1 in alveolar rhabdomyosarcoma cells (Fig. 2A), thapsigargin also decreased PAX3-FOXO1 protein levels in these cells (Fig. 1E). It was postulated that the AKT activation–mediated increase in phosphorylated PAX3-FOXO1 will subsequently be declined in alveolar rhabdomyosarcoma cells treated with thapsigargin. Indeed, an increased level of phosphorylated PAX3-FOXO1, followed by decreased protein levels, was observed in thapsigargin-treated Rh30 cells (Fig. 3D). The increased level of phosphorylated PAX3-FOXO1 was further correlated with AKT activation in thapsigargin-treated cells. Therefore, the mechanism that decreases PAX3-FOXO1 protein levels in thapsigargin-treated alveolar rhabdomyosarcoma cells was examined. Studies showed that thapsigargin induces the ER stress response pathways leading to proteasome-dependent degradation of proteins (34). To evaluate whether ER stress signals were related to the decreased PAX3-FOXO1 protein levels, Rh28 and Rh30 cells were treated with dithiothreitol (DTT), which causes ER stress by interfering with disulfide bond stability (35, 36), and analyzed by immunoblot analysis. A reduced level of PAX3-FOXO1 protein was observed in cells treated with thapsigargin as anticipated, but not with DTT (Supplementary Fig. S3), suggesting that the decreased PAX3-FOXO1 protein levels may be caused by proteosomal degradation independent of ER stress signals. Therefore, PAX3-FOXO1 protein levels were evaluated in Rh30 cells by incubating with thapsigargin alone or co-incubating with proteosomal inhibitor MG132. The data showed that the decreased PAX3-FOXO1 protein levels in thapsigargin-treated cells were blocked by MG132 (Fig. 3E), showing its proteasomal-dependent degradation. The data presented thus far suggest that activation of AKT by thapsigargin promotes PAX3-FOXO1 phosphorylation, which leads to its proteasomal degradation. Therefore, the effect of AKT activation on PAX3-FOXO1 protein levels was examined in Rh30 cells that have transduced with retrovirus-expressing myrAKT-HA. As shown in Fig. 3F, PAX3-FOXO1 protein was practically absent in cells that expressed hemagglutinin (HA)-tagged myrAKT. Moreover, the absence of PAX3-FOXO1 protein was associated with decreased expression of its target MyoD in these cells. Together, these results suggest that thapsigargin elevates AKT-stimulated phosphorylation of PAX3-FOXO1 and subsequently leads to its proteasomal degradation.
Thapsigargin inhibits PAX3-FOXO1 binding on the regulatory elements of its target genes
Because thapsigargin inhibits PAX3-FOXO1–driven gene transcription in alveolar rhabdomyosarcoma cells (Fig. 2B), it may be resulted from thapsigargin-induced inhibition of PAX3-FOXO1 binding to the regulatory regions of its target genes. Hence, the effect of thapsigargin on such PAX3-FOXO1 binding to its targets was decided to conduct in human alveolar rhabdomyosarcoma cells by chromatin immunoprecipitation (ChIP) assay. Genome-wide binding study showed that PAX3-FOXO1–binding sites are enriched with PAX3 motifs and strongly associated with induced expression of genes in human alveolar rhabdomyosarcoma cells (29). Although human alveolar rhabdomyosarcoma cells express low levels of wild-type PAX3 (ref. 26; Supplementary Fig. S1B), which constitutes the N-terminal DNA–binding side of PAX3-FOXO1, the use of PAX3-specific antibodies to examine thapsigargin-induced effects specifically on PAX3-FOXO1 binding by ChIP assay was avoided in these cells. The rationale was that it may produce a wild-type PAX3-specific background noise for such PAX3-FOXO1 binding by ChIP in human alveolar rhabdomyosarcoma cells. Moreover, the use of FOXO1-specific antibodies is avoided for the above ChIP assay because PAX3-FOXO1 binding to its target genes is not contributed by the DNA-binding domain of wild-type FOXO1 (37). However, PAX3-FOXO1–specific antibodies are not yet commercially available. Therefore, a PAX3-FOXO1–binding study was conducted in human Rh30 alveolar rhabdomyosarcoma cells following the overexpression of HA epitope–tagged PAX3-FOXO1 and using HA antibodies for ChIP. As shown in Fig. 4A, an increased level of PAX3-FOXO1 along with the expression of HA protein was observed in Rh30 cells transduced with lentivirus-expressing PAX3-FOXO1-HA. These were labeled as Rh30-PAX3-FOXO1-HA cells and subsequently used for ChIP to evaluate the effect of thapsigargin on PAX3-FOXO1 binding to the core enhancer regulatory region of its known target MYOD (29). A reduction in PAX3-FOXO1 binding to the enhancer region of MYOD was observed in cells incubated with thapsigargin (Fig. 4B). Moreover, the analysis of chromatin used for ChIP showed that the expression of PAX3-FOXO1-HA was not affected by thapsigargin, suggesting that the deficiency of PAX3-FOXO1 chromatin occupancy on MYOD was not due to the decreased levels of PAX3-FOXO1 protein in thapsigargin-treated cells. The quantitative PCR analysis of ChIP DNA also showed that PAX3-FOXO1 chromatin occupancy on MYOD and second intron of IGF1R, which is a target bound and directly regulated by PAX3-FOXO1 (29), was severely impaired in thapsigargin-treated cells (Fig. 4C). Together, the data imply that thapsigargin eradicates PAX3-FOXO1 binding to its targets resulting in the suppression of PAX3-FOXO1–mediated gene transcription in alveolar rhabdomyosarcoma cells.
Thapsigargin inhibits alveolar rhabdomyosarcoma cell tumorigenic potential and induces apoptosis in vitro
Studies have indicated that the inhibition of either PAX3-FOXO1 activity or expression blocks the growth and induces apoptosis of alveolar rhabdomyosarcoma cells (14, 18, 20, 31). As thapsigargin suppresses PAX3-FOXO1 activity and subsequently its abolition in alveolar rhabdomyosarcoma cells (Fig. 2 and 3), the effect of thapsigargin on alveolar rhabdomyosarcoma cell growth was examined. The results showed that thapsigargin severely impaired the growth of a panel of alveolar rhabdomyosarcoma cell lines (Fig. 5A). The effectiveness of thapsigargin on the survival ability of these alveolar rhabdomyosarcoma cells to grow into colonies was also examined and the data showed a severe decrease in number of colonies in thapsigargin-treated cells (Fig. 5B). These results provoked the exploration of apoptosis in thapsigargin-treated alveolar rhabdomyosarcoma cells by examining the levels of cleaved caspase-3 and PARP, which are biomarkers of apoptosis. Indeed, an increased level of these apoptotic markers was observed in Rh28 and Rh30 cells treated with thapsigargin (Fig. 5C). Moreover, thapsigargin-induced apoptosis in these cells was evident by detecting apoptotic DNA fragmentation, a key biochemical hallmark of apoptosis (Supplementary Fig. S4).
Studies have also implicated that PAX3-FOXO1 expression in alveolar rhabdomyosarcoma confers an aggressive phenotype in vivo such as tumorigenic and metastatic potential (5, 38). The anchorage-independent growth of tumor cells in vitro is generally assumed to be closely related to the above in vivo events. Therefore, the effect of thapsigargin on the ability of alveolar rhabdomyosarcoma cells to exhibit anchorage-independent cell growth was evaluated in Rh30 and U20325 cells by examining colony-forming capacity in semisolid soft agar media. The results showed that thapsigargin inhibited the growth of these cells as evidenced by the decreased number of colonies (Fig. 5D). In addition, the effect of thapsigargin was evaluated on invasive behavior of alveolar rhabdomyosarcoma cells, one of the hallmarks of the metastatic potential. This was conducted by treating Rh30 and U20325 cells with thapsigargin and measuring the invasiveness with a Matrigel invasion assay. The data showed that thapsigargin also inhibited these cells invasion through Matrigel (Fig. 5E). Together, these in vitro results show that thapsigargin is able to block alveolar rhabdomyosarcoma cell growth, survival, metastatic ability and induce apoptosis.
Thapsigargin inhibits the growth of human alveolar rhabdomyosarcoma xenografts in vivo
Finally, the in vivo effect of thapsigargin on tumor growth was evaluated using an Rh28 alveolar rhabdomyosarcoma xenograft mouse model. Initial dose finding experiment in wild-type mice showed the maximum tolerable single intravenous dose of thapsigargin, which did not produce mortality, was 0.2 mg/kg body weight. Subsequently, Rh28 xenografts were treated with thapsigargin (single administration) at 2 different doses (0.1 and 0.15 mg/kg); control mice received a one-time PBS treatment and tumor growth was measured. As anticipated, neither of the above one-time dosing regimens of thapsigargin produced any significant changes in body weight from treatment to the time of euthanization (Fig. 6A). However, the mice that were treated with thapsigargin either 0.1 or 0.15 mg/kg showed a significant reduced tumor growth when measuring the tumor volume (Fig. 6B). To further characterize the effect of thapsigargin on tumor growth in vivo, the resected tumors from both thapsigargin-treated and control mice were sectioned and stained with H&E or used for immunohistochemical analysis. As shown in Fig. 6C, H&E staining of tumor sections showed less viable round cell morphology in thapsigargin-treated mice (Fig. 6C). Moreover, tumors sections stained with antibody against proliferation marker Ki-67 and apoptosis-inducing activated caspase-3 evidently showed the decreased Ki-67 but increased activated caspase-3–positive cells in thapsigargin-treated mice. Together, the results display in vivo inhibition of tumor cell proliferation and concomitant increased apoptosis in alveolar rhabdomyosarcoma tumor model following thapsigargin treatment.
In this study, a cell-based screening system was used to identify small molecule inhibitors of PAX3-FOXO1, a unique fusion oncogenic transcription factor present only in childhood cancer alveolar rhabdomyosarcoma (5, 17). The system constituted a PAX3-FOXO1–responsive reporter gene in PAX3-deficient alveolar rhabdomyosarcoma cells expressing endogenous PAX3-FOXO1 protein to screen a library of 3,280 compounds for inhibitors of PAX3-FOXO1 transcriptional activity. This system was an important simplification on measuring transcriptional activity solely mediated by PAX3-FOXO1, which successfully identified 3 lead compounds that inhibited PAX3-FOXO1 activity. The most promising compound identified was the SERCA inhibitor thapsigargin. This compound was then evaluated for its potential as an inhibitor of PAX3-FOXO1. The results showed that thapsigargin elevates AKT-coupled phosphorylation of PAX3-FOXO1, resulting in the suppression of its activity and subsequently proteosomal degradation in alveolar rhabdomyosarcoma cells. Moreover, thapsigargin inhibited the alveolar rhabdomyosarcoma cells growth and invasiveness and induced the apoptosis of these cells in vitro. Most importantly, thapsigargin suppressed the growth of alveolar rhabdomyosarcoma xenografts in vivo. These results underscore the robustness of this cell-based system in identifying thapsigargin as a first-in-class inhibitor of PAX3-FOXO1 suppressing alveolar rhabdomyosarcoma cell growth and malignant phenotypes. Moreover, this system can be expanded in evaluating additional small molecule libraries to identify potential inhibitors of PAX3-FOXO1.
The screening-identified thapsigargin was originally isolated from plant Thapsia garganica L. (Linnaeus; ref. 39). Although thapsigargin is most widely used inhibitor of SERCA to study intracellular Ca2+ signaling (21), its action is also associated with the activation of apoptosis in cells (33). This apoptotic aspect of thapsigargin has recently exploited to develop a thapsigargin-derived prodrug for cancer therapy (40). First and foremost, this study uncovered that thapsigargin inhibits PAX3-FOXO1 transcriptional activity as shown by suppression of its driven reporter gene transcription and expression of downstream targets MyoD and SKP2 in alveolar rhabdomyosarcoma cells. Studies have reported thapsigargin-induced activation of multiple kinase signaling pathways (41), including activation of AKT (32). In alveolar rhabdomyosarcoma cells, AKT activation also results in PAX3-FOXO1 phosphorylation–coupled decreased transcriptional activity (23). The data in this study reveal that thapsigargin inhibited PAX3-FOXO1 transcriptional activity is concurrent with AKT activation leading to PAX3-FOXO1 phosphorylation in alveolar rhabdomyosarcoma cells. In support of the above view is that AKT inhibitor MK-2206 overcomes thapsigargin-inhibited PAX3-FOXO1–responsive gene transcription. Although the increase of intracellular calcium levels by thapsigargin accounts for its action, most stunning in this esteem is that CaCl2 alone increased AKT activation alongside with decreased PAX3-FOXO1–responsive gene transcription. As thapsigargin-induced AKT activation is downstream of calcium release (32), the findings of CaCl2 suggest that thapsigargin-induced suppression of PAX3-FOXO1 activity results from activation of AKT via Ca2+-induced signaling pathways. Currently, it is unclear how thapsigargin/Ca2+ activates AKT in alveolar rhabdomyosarcoma cells and experiments are aimed at elucidating the mechanisms of this activation.
In alveolar rhabdomyosarcoma cells, PAX3-FOXO1 phosphorylation either by PKC or GSK3 positively regulate its transcriptional activity (18, 31) and the blockade of phosphorylation on PAX3 domain of PAX3-FOXO1 results in its binding incompetence to targets, thereby transcriptional inactivation (18). In thapsigargin-treated alveolar rhabdomyosarcoma cells, however, the data show that AKT activation–mediated inhibition of PAX3-FOXO1 activity via phosphorylation impairs its binding to the target genes. Because PAX3-FOXO1 contains 2 consensus AKT phosphorylation sites in the FOXO1 domain (42), thapsigargin-inhibited binding of PAX3-FOXO1 on its target genes may be resulted from the phosphorylation of these AKT sites. In that case, it may be concluded that phosphorylation of AKT sites in PAX3-FOXO1 impedes its binding to target gene transcription. This inhibition of PAX3-FOXO1 binding by thapsigargin may arise as a consequence of AKT activation mediating a blockade of downstream effector GSK3, which has been implicated in PAX3-FOXO1 phosphorylation and subsequent transactivation (31). Experiments are now underway in assessing these scenarios in thapsigargin-treated alveolar rhabdomyosarcoma cells.
It has been shown that the inhibition of PAX3-FOXO1 phosphorylation blocks its transcriptional activity alongside with increased protein levels in alveolar rhabdomyosarcoma cells (18). However, the data presented here show that thapsigargin-induced transcriptional inactive phosphorylated PAX3-FOXO1 protein subsequently declines in these cells. It seems that there is a phosphorylation-dependent transcriptionally inactive PAX3-FOXO1 degradation in thapsigargin-treated alveolar rhabdomyosarcoma cells. The ER stress inducer thapsigargin triggers proteasome-dependent degradation of cellular proteins (43). Although pharmacologic blockade of proteasomal activity inhibits the decline of PAX3-FOXO1 protein in thapsigargin-treated alveolar rhabdomyosarcoma cells, it can be argued against thapsigargin-induced proteasomal activity in this scenario. Supporting evidence is the inability of DTT, a known ER stress inducer, to decrease PAX3-FOXO1 protein levels in alveolar rhabdomyosarcoma cells. It was hypothesized that thapsigargin-induced AKT activation might trigger proteasome-dependent degradation of phosphorylated PAX3-FOXO1 in these cells. This is supported by the results that show deceased levels of PAX3-FOXO1 protein in alveolar rhabdomyosarcoma cells expressing a constitutively active form of AKT. Taken together, the data in this study offer that the regulation of PAX3-FOXO1 activity and expression by AKT such that phosphorylation blocks activity and promotes degradation of PAX3-FOXO1. An emphasis to this view is the therapeutic significance of thapsigargin in the inhibition of PAX3-FOXO1 activity and expression in alveolar rhabdomyosarcoma cells. This was predicted on the basis of studies that have shown the persistent dependence of alveolar rhabdomyosarcoma cells on the presence of PAX3-FOXO1, as its downregulation reduces growth and motility and induces apoptosis (9, 12–14, 44). Indeed, the data presented here show that thapsigargin is effective in inhibiting cell growth and invasion and inducing apoptosis of alveolar rhabdomyosarcoma cells in vitro. Most importantly, thapsigargin is effective in reducing tumor growth in an Rh28 alveolar rhabdomyosarcoma xenograft in vivo.
In summary, this study identifies thapsigargin as an inhibitor of PAX3-FOXO1 transcriptional activity and expression in alveolar rhabdomyosarcoma cells. Moreover, the findings highlight the significance of thapsigargin-induced AKT activation in the alteration of PAX3-FOXO1 function resulted inhibition of alveolar rhabdomyosarcoma. Central to this perspective is the finding that thapsigargin-induced Ca2+-dependent AKT activation, via unknown mechanisms, leads to PAX3-FOXO1 phosphorylation and inhibition of its binding to suppress target gene expression and subsequent proteasomal degradation. Such thapsigargin actions on PAX3-FOXO1 eventually facilitate the block of alveolar rhabdomyosarcoma. On the basis of these data, we conclude that thapsigargin could be developed as a potential therapeutic agent for such and possibly related translocation-positive childhood alveolar rhabdomyosarcoma.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Conception and design: M. Jothi, A.K. Mal
Development of methodology: M. Jothi, M. Mal, A.K. Mal
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): M. Jothi
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): M. Jothi
Writing, review, and/or revision of the manuscript: M. Jothi, C. Keller, A.K. Mal
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): M. Jothi
Study supervision: A.K. Mal
Provision of primary cell cultures: C. Keller
The authors thank the Small Molecule Screening Core, Pathology Resource Network and Animal Core (Roswell Park Cancer Institute), Jean Veith for intravenous injection into mice. They also thank Dr. Norman J. Karin and David W. Wolff for critical suggestions and editorial support.
This work was supported by Public Health Service grant AR051502 from National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) and Roswell Park Alliance Foundation grant to A.K. Mal.
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